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Cardiovascular Research 2003 59(2):380-389; doi:10.1016/S0008-6363(03)00429-2
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Copyright © 2003, European Society of Cardiology

Transgenic rat hearts overexpressing SERCA2a show improved contractility under baseline conditions and pressure overload

Oliver J. Müllera, Mathias Langeb, Henning Rattundeb, Hans-Peter Lorenzenb, Matthias Müllerb, Norbert Freya, Cordula Bittnerc, Warner Simonidesd, Hugo A. Katusa and Wolfgang-M. Franze,*

aInnere Medizin III, Universitätsklinikum Heidelberg, Heidelberg, Germany
bMedizinische Klinik II, University of Lübeck, Lübeck, Germany
cDepartment of Pathology, University of Lübeck, Germany
dLaboratory for Physiology, VU University Medical Center, Amsterdam, The Netherlands
eMed. Klinik und Poliklinik I—Großhadern, Klinikum der Universität München, Marchioninistr. 15, D-81377 Munich, Germany

* Corresponding author. Tel.: +49-89-7095-3094; fax: +49-89-7095-6094. wfranz{at}helios.med.uni-muenchen.de

Received 27 November 2002; accepted 7 April 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA) is reduced in the failing myocardium. Therefore, transfer of SERCA2a cDNA is considered as a therapeutical approach. The aim of this study was analysis of the long-term effect of SERCA2a overexpression in normal as well as pressure overload challenged myocardium of transgenic rats. Methods: Independent transgenic rat lines were established expressing the rat SERCA2a cDNA specifically in the myocardium resulting in increased SERCA2a protein levels by 30–70%. Simultaneous measurements of isometric contraction and calcium transients were carried out in right ventricular papillary muscle preparations. Hemodynamic parameters were measured in hearts of unchallenged rats as well as 10 weeks after pressure overload induced by abdominal aortic banding. Results: Analysis of calcium handling and contractile parameters in isolated right ventricular papillary muscles revealed significant shortening of intracellular calcium transients and half maximal relaxation times (RT50). Assessing myocardial contractility in working heart preparations, both transgenic rat lines revealed elevated left ventricular pressure, improved systolic and diastolic parameters, attenuated negative force–frequency relation, and a dose-dependent β-adrenergic effect. Aortic banding resulted in reduction of left ventricular pressure and worsening of contraction and relaxation parameters with no differences in mortality in both transgenic (+dP/dt 3084±96 vs. 3938±250 mmHg/s; RT50 47.0±1.2 vs. 36.7±1.4 ms) and wild-type rats (+dP/dt 2695±86 vs. 3297±122 mmHg/s; RT50 53.0±1.6 vs. 44.1±1.4). SERCA2a overexpressing hearts revealed improved hemodynamic parameters compared to wild-type controls. Acceleration of isovolumetric relaxation characterized by the index Tau was directly correlated to SERCA2a protein concentrations. Conclusion: Overexpression of SERCA2a protein results in a positive inotropic effect under baseline conditions remaining preserved under pressure overload without affecting mortality. Therefore therapeutic transfer of SERCA2a may become a potential approach for gene therapy of congestive heart failure. Moreover, transgenic SERCA2a rats will be useful for studies of long-term SERCA2a overexpression in further cardiovascular disease models.

KEYWORDS Ca-pump; Contractile function; Gene therapy; Heart failure; SR (function)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Human heart failure is characterized by decreased systolic Ca2+ release from the sarcoplasmic reticulum (SR) and increased diastolic Ca2+ concentrations in the cardiomyocyte [1]. These alterations of intracellular calcium homeostasis have been attributed to a decreased Ca2+ reuptake into the sarcoplasmic reticulum during diastole [2]. In mammalian myocardium, the SR Ca2+-ATPase (SERCA) is responsible for most of the Ca2+ removal during relaxation. This pump transports two calcium molecules from the cytoplasmic compartment into the sarcoplasmic reticulum (SR) by an energy consuming process. Previous studies have shown that the activity of SERCA is decreased in failing myocardium [2]. Whether this can be attributed to a potential downregulation of SERCA at the protein level has not been fully resolved [2,3]. Downregulation of SERCA2a also occurs in patients with hypertrophic cardiomyopathy and was associated with diastolic dysfunction [4].

The effect of transient SERCA2a overexpression was previously studied by adenoviral transfer of SERCA2a in a rat model of heart failure induced by constriction of the ascending aorta [5]. Overexpression of SERCA2a restored ventricular function to levels comparable to sham-operated controls within 2–3 days. More importantly, adenoviral SERCA2a transfer increased survival after 4 weeks without adverse effects on energy metabolism [6]. Since adenoviral transfer is transient, there was no information on chronic SERCA2a overexpression in this study.

Previous transgenic mouse models overexpressing SERCA2a in the heart have provided valuable insight into the role of SERCA enhancing calcium transients as well as contraction and relaxation parameters [7,8]. Although precise hemodynamic measurements can be carried out in mice [9,10], the use of transgenic mouse models is hampered since few pathophysiological models have been established in mice [11]. Furthermore, the murine cardiovascular system has several unique characteristics [12]. For example, the murine myocardium is less sensitive to Ca2+ when compared to rats or other mammalians. Therefore, a lower Ca2+ tolerance in larger mammals could result in long-term side-effects which may not be observed in mice. Moreover, mechanisms involved in enhancing cardiac systolic function such as force–frequency relationship or adrenergic stimulation may play only a minor role in mice [10,13]. Finally, the phenotype of both transgenic mouse models overexpressing SERCA2a may be influenced by the usage of SERCA2a cDNA of another species.

Therefore we established independent transgenic rat lines expressing SERCA2a cDNA of the same species. This model enables analysis of a long-term SERCA2a overexpression on calcium transients, contraction and relaxation parameters of unchallenged as well as pressure overloaded hearts.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Microinjection construct
SERCA2a cDNA was excised from pBS-SERCA (gift from Dr K. Boheler, National Institute on Aging, Baltimore, MD, USA) [14] and linked to the ventricular specific rat 2.1 kb myosin light chain-2v (MLC-2v) promoter [15]. At the 3'-end, the SV40 untranslated region (EcoNI/SalI) of the pGL2 vector (Promega, USA) was introduced via NotI. At the 5'-end of the MLC-2v promoter, an oligonucleotide containing an EcoRV and a NotI-site was inserted via KpnI. The newly generated NotI site was used for introduction of the –590/91-bp fragment of the human cytomegalovirus (CMV) immediate-early enhancer to increase expression levels [16,17]. The microinjection construct was linearized by restriction with BssHII yielding a fragment of 8 kb (Fig. 1a).


Figure 1
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  		Fig. 1 Generation and characterization of transgenic rat lines. (A) Microinjection construct for cardiac-specific overexpression of rat 3.9 kb SERCA2a cDNA under control of rat 2.1 kb MLC-2v promoter and 0.5 kb CMV-IE enhancer. (B) Western blot analysis showing expression of SERCA2a in wild-type (–/–) and homozygous (+/+) transgenic rat lines 104.1 and 104.2. For densitometric quantification of transgene expression, cardiac troponin T was used for standardization. (C) Western blot analysis showing expression of PLB in wild-type (–/–) and the homozygous (+/+) high expressing SERCA2a line 104.1 in relation to cardiac troponin T. CMV-IE: cytomegalovirus immediately-early; MLC-2v: ventricular myosin light chain-2; SV40: simian virus 40.

 
2.2 Generation and analysis of transgenic rats
Transgenic rats were generated according to procedures published previously [18]. DNA extracted from tail biopsies of offspring was analyzed for transgene integration by PCR (primers MLCSER 5'-CCTCACCTACAACTGCCAAAAG-3'; SER5 5'-AACCAAGCCAAAACGAAAGATA-3') and Southern blotting using a Digoxigenin-labeled probe (Roche, Germany).

2.3 Animal procedures
All experiments conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Animals were anesthetized with ether and sacrificed by dislocation of the neck. After thoracotomy, hearts were rapidly excised.

For abdominal aortic banding, transgenic and wild-type rats of 8–10 weeks were anesthetized with pentobarbital sodium (Nembutal; 375 mg/100 g i.p.). To induce pressure overload by both outflow constriction and Goldblatt mechanism, the abdominal aorta was constricted between the renal arteries [19–21]. For standardization, a 4.0 silk suture was tied around the abdominal aorta and a blunted 22-gauge needle, which was removed afterwards. Ten weeks later, the rats were sacrificed and hearts excised for working heart analyses. Successful banding was confirmed by hypoplasia of the ischemic kidney.

2.4 Protein analysis
Rat tissue was immediately frozen in liquid nitrogen. Then, 10-µg aliquots of total protein homogenate were analyzed by Western blotting using a polyclonal SERCA2a antibody (1:8000) [22], a monoclonal phospholamban (clone A1) antibody (1:1000) (Upstate Biotechnology, USA) and a monoclonal troponin T (1H10 [PDB] ) antibody (1:50 000) [23]. Detection of primary antibodies was carried out with the ECL-kit (Amersham, UK) using peroxidase-labelled secondary antibodies against mouse (1:10 000) and rabbit IgG (1:2000) (DAKO, Hamburg, Germany), respectively. Signals were quantified by transmission densitometry. For standardization, values of the SERCA2a and phospholamban signals were divided by the corresponding cardiac troponin T signal detected with a monoclonal antibody as previously described [17]. SERCA protein levels were additionally determined immunochemically by dot-blot analysis, as described before, with minor modifications [24]. Briefly, homogenate tissue samples (typically 0.5 µg total protein) and standard SERCA2 protein samples were spotted onto a nitrocellulose membrane. The blot was then incubated with a 1:2500 dilution of a polyclonal antiserum to SERCA2a [22], and subsequently with 125I-labeled anti-rabbit immunoglobulin G (0.05 µg/ml, specific activity 7 µCi/µg). Blots were exposed to Phosphor Imager screens, which were then scanned and spots were quantified using ImageQuant software (Molecular Dynamics).

2.5 Determination of calcium transients and isometric contractility in papillary muscles
Rat hearts were incubated for a period of 10 min in a cardioplegic carbogenated Krebs–Henseleit solution containing 30 mM 2,3-butanedione-2-monoxime (BDM). Right ventricular papillary muscles (length 2–3 mm; cross-section 0.5 mm2) were excised and incubated in a carbogenated Krebs–Henseleit solution with addition of 5 µM fura-2-acetoxymethylester, tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (0.43 mg/100 ml) and 0.75% cremophor for 3 h. Consecutively papillary muscles were mounted between two tweezers in a chamber filled with Krebs–Henseleit buffer at room temperature. One of the tweezers was connected to an isometric force transducer (BAM4c, Scientific instruments, Heidelberg, Germany) and forces were recorded [25]. Muscles were stretched for 30–60 min to the length at which active force development was maximal and stimulated with 1 Hz. Relaxation time (RT50) was determined as the time from the peak of contraction to 50% of maximum developed stress during relaxation. For measurements of calcium transients, the muscle preparation was illuminated at alternating excitation wavelengths of 340 nm (calcium dependent) and 380 nm (calcium independent). Images of 340 nm were divided by those at 380 nm to determine the fluorescence ratio correlating with the intracytoplasmatic calcium concentration.

2.6 Isolated working heart perfusions
Myocardial function was evaluated using isolated working heart preparations [26]. Hearts were rapidly excised, mounted at the perfusion apparatus (Type 844, Hugo Sachs Elektronik, Germany) and perfused in a retrograde non-recirculating Langendorff technique with a modified Krebs–Henseleit buffer [17].

The left atrium was cannulated to allow a perfusion via the left atrium according to the working heart technique of Neely et al. [26]. Left atrial pressure and aortic pressure were determined with an Isotec pressure transducer (Hugo Sachs Elektronik KG, March, Germany). For measurement of intraventricular pressures, a Millar-tip catheter was inserted in the left ventricle via the aortic valve. After a 15-min stabilization period of Langendorff perfusion, hearts were switched to the working heart mode. A preload of 8–10 mmHg and an afterload of 80 mmHg was used to ensure an adequate coronary flow. Hearts were electrically paced by means of an electrode (Hugo Sachs stimulator T) near the sinoatrial node at a constant rate of 300 beats per min (bpm) for the baseline protocol. In addition, hemodynamic parameters were measured at increasing heart rates up to 375 bpm and continuous infusions of increasing isoproterenol concentrations from 3x10–9 to 3x10–6 mol/l.

Data on contractile performance were collected on-line at a sampling rate of 100 Hz with an electronic data acquisition system (MacLab ADInstruments, USA). The IGOR Pro 3.0 software (Wisstech, Oberhausen, Germany) was used to calculate contraction and relaxation parameters based on the average of 10 sequential beats.

2.7 Histological analyses
After dissection, hearts were washed in PBS, fixed, and paraffin-embedded for sectioning. Routine staining was carried out with hematoxylin–eosin. The individual width of the myocytes was quantified microscopically by a pathologist blinded to the genotype of the animal (Zeiss, Germany).

2.8 Statistical analyses
All data were expressed as mean±S.D. To test for statistical difference in heart to body weight, an unpaired Student's t-test (level of significance <0.05) was applied. To examine the effects of pacer stimulation and isoproterenol application on hemodynamic changes, a two-way repeated measurement analysis of variance (ANOVA) was used.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Generation and characterization of transgenic rats overexpressing SERCA2a
To evaluate a long-term effect of SERCA2a overexpression on cardiac function, transgenic rats were generated by microinjection of the 8.0 kb CMV-MLC2v-SERCA2a-SV40polyA fusion gene (Fig. 1A) into the male pronuclei of fertilized rat oocytes. PCR and Southern blot analyses of DNA extracted from tail biopsies identified germline integration in two independent rat lines. After breeding to homozygosity both lines (104.1 and 104.2) were analyzed for SERCA2a expression in relation to cardiac troponin T which is stable expressed. Western blot analyses showed a significant increase in SERCA2a protein expression of 1.7- (line 104.1) and 1.3- (line 104.2) fold compared to their wild-type littermates (Fig. 1B). Phospholamban (PLB) protein levels of transgenic SERCA2a lines revealed no significant change compared to wild-type hearts (Fig. 1C). Although there was a trend towards an increase in heart to body weight ratio, differences were not significant compared to wild-type (Table 1). No mortality was observed in the groups of sham operated and banded wild-type and transgenic rats. Histological analyses revealed similar values for wall thickness and myocyte diameters.


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  		Table 1 Parameters in isolated work-performing rat heart preparations of SERCA2a transgenic and wild-type-rats

 
3.2 Analyses of intracellular calcium transients and half maximal relaxation times in right ventricular papillary muscles of SERCA2a overexpressing rats
To determine the influence of SERCA2a overexpression on intracellular calcium homeostasis, transgenic and non-transgenic papillary muscles were analyzed for calcium transients under conditions of isometric contraction after loading with fura-2. Right ventricular papillary muscles from transgenic and non-transgenic controls were used because of a better oxygenation due to the small mean cross-sectional area of 0.5 mm2. Isometric force development at optimal length and time to peak (TTP) pressure was not significantly different between transgenic and wild-type muscles. However, papillary muscles derived from high expressing SERCA2a line 104.1 revealed a trend to develop higher force (14.4±0.8 mN/mm2) compared to wild-type force (11.0±0.6 mN/mm2) in a shorter time. Half-maximal relaxation time (RT50) was decreased in both transgenic lines (Fig. 2A), which was statistically significant only for line 104.1. Shortening of RT50 was paralleled by a significant acceleration of intracellular calcium transients in both transgenic lines (Fig. 2B). In summary, overexpression of SERCA2a resulted in an accelerated relaxation associated with an increased decline of intracellular calcium concentration in right ventricular papillary muscle.


Figure 2
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  		Fig. 2 Simultaneous measurements of isometric contraction and calcium transient in right ventricular papillary muscles of SERCA2a transgenic lines. Each graph is based on the average of 50 twitches per experiment in five to six independent papillary muscle preparations. (A) Relative isometric force development at optimal length of right ventricular papillary muscles of SERCA2a transgenic lines and wild-type controls. The half maximal relaxation time (RT50) of transgenic lines was significantly shorter (109±5 ms*) in line 104.1 compared to wild-type (134±5 ms) indicating an accelerated relaxation. (B) Calcium transients were determined simultaneously using fura-2 loaded papillary muscles contracting isometrically at optimal length. The half maximal relaxation time (RT50) was significantly shorter in transgenic (163±7 ms* in line 104.2 and 173±5 ms* in line 104.1) compared to wild-type rats (238±5 ms) suggesting a faster calcium reuptake into the sarcoplasmic reticulum. *P<0.05; values represent mean±S.E.M.

 
Diastolic force levels are an important indicator for the contractile status of isolated heart muscles. Therefore, we have evaluated diastolic force at the basal stimulation frequency of 1 Hz and found no difference between transgenic and wild-type muscles (3.7±1.7 vs. 2.9±2.1 mN/mm2; P = n.s.). Since diastolic force itself depends on diastolic intracellular Ca levels, we also measured fura-2 ratio (340 nm/380 nm) and found no difference at 1 Hz between transgenic and wild-type preparations (1937±379 vs. 1659±337; P = n.s.).

3.3 Characterization of SERCA2a overexpressing rats in the work performing heart model
Functional consequences of SERCA2a overexpression in homozygous transgenic rats were analyzed in isolated hearts by the working heart perfusion apparatus. Baseline measurements were made at constant preload (8–10 mmHg), afterload (80 mmHg) and heart rate (300 bpm). Systolic intraventricular pressure (IVP) was significantly elevated in transgenic rats compared to wild-type (Table 1). Systolic function represented by TTP and maximum systolic pressure rise (+dP/dt) was also significantly increased. Relaxation parameters represented by the maximum diastolic pressure decline (–dP/dt), RT50, and RT90 were significantly shortened. The preload independent parameter of isovolumetric relaxation, Tau [27], was also significantly reduced in line 104.1. Analysis of a further line (104.2) revealed accelerated contraction and relaxation parameters similar to line 104.1 (data not shown). However, alterations in line 104.1 were more pronounced compared to 104.2 which might reflect the higher level of SERCA2a overexpression.

Furthermore, the effect of SERCA2a-overexpression on hemodynamic parameters was analyzed under different stimulatory frequencies. An increase in heart rate from 300 to 375 min–1 led to a fall in systolic left ventricular pressure in both transgenic and wild-type rats (Fig. 3A). In contrast to wild-type hearts which showed an increase in Tau from 14.9±0.8 to 18.8±2.7 ms, transgenic SERCA2a hearts revealed no significant change (Fig. 3B). In transgenic line 104.1, Tau was significantly lowered in comparison to wild-type at all stimulation frequencies, indicating that the phase of active relaxation remains accelerated over the range of physiological heart rate.


Figure 3
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  		Fig. 3 Pacemaker stimulation of isolated work-performing heart preparations of SERCA2a transgenic and wild-type rats. (A) Effect of increasing heart rates on intraventricular pressure (IVP). An increase in the heart rate from 300 to 375 min–1 led to a significant decrease in IVP in transgenic and control hearts. (B) Influence of increasing heart rates on the preload independent index of isovolumetric relaxation, Tau. In transgenic line 104.1 Tau was significantly decreased compared to wild-type in all measurements. Each data point represents 10–12 independent experiments. At least 10 sequential beats were averaged for each experiment. *P<0.05; **P<0.01; values represent mean±S.E.M.

 
In order to analyze whether contraction and relaxation parameters can be further accelerated in transgenic SERCA2a rats, isolated hearts were subjected to increasing doses of isoproterenol. In wild-type hearts, IVP rose from 96±3 to 157±8 mmHg (P≤0.05) at maximal doses of isoproterenol. In line 104.1, IVP rose from 127±7 to 198±13 mmHg (P≤0.05) and in line 104.2 from 118±4 to 193±14 mmHg (P≤0.05) (Fig. 4A). Contraction and relaxation velocities (+dP/dt and –dP/dt) were not significantly different in hearts from both transgenic lines and wild-type controls with increasing doses of isoproterenol (data not shown). In contrast, Tau was significantly shortened in line 104.1 compared to wild-type even at the maximal dose of isoproterenol (Fig. 4B). These data indicate that SERCA2a transgenic rat hearts can be stimulated by β-agonists resulting in a dose-dependent elevation of systolic IVP paralleled by an accelerated isovolumetric relaxation.


Figure 4
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  		Fig. 4 Isoproterenol stimulation of isolated work-performing heart preparations of SERCA2a transgenic and wild-type rats. (A) Effect of isoproterenol on intraventricular pressure (IVP). Increasing isoproterenol concentrations up to 3x10–6 mol/l led to a rise of IVP in transgenic as well as control hearts. In transgenic line 104.1, IVP was significantly higher compared to wild-type in all measurements. (B) Influence of isoproterenol on the preload independent index of isovolumetric relaxation, Tau. In wild-type and transgenic hearts Tau decreased with rising isoproterenol concentrations. In transgenic line 104.1, Tau was significantly lower compared to wild-type in all measurements. Each data point represents 10–12 independent experiments. Ten sequential beats were averaged for each experiment. *P<0.05; **P<0.01; values represent mean±S.E.M.

 
3.4 Characterization of SERCA2a overexpressing rats after banding of the abdominal aorta
Transgenic rats of line 104.1 and wild-type rats were challenged by constriction of the abdominal aorta between the renal arteries resulting in long-term pressure overload by both outflow constriction and Goldblatt mechanism [19,20]. Successful banding was confirmed demonstrating hypoplasia of the ischemic kidney. The effect of pressure overload on physiological parameters was analyzed 10 weeks after surgery. No mortality was observed in the groups of sham operated and banded wild-type and transgenic rats. The hypertrophic effect of the banding procedure was reflected in a significant increase in heart/body weight ratio in both banded SERCA2a and wild-type rats (Table 1) as well as a trend towards increased myocyte diameters (data not shown). Hemodynamic parameters of banded animals analyzed in isolated working heart preparations revealed a decrease in IVP and systolic function reflected by +dP/dt (Table 1). However, there was no change in TTP in transgenic rats, which may be due to a concordant decline in the IVP and +dP/dt. Diastolic function reflected by –dP/dt, RT50 and RT90 was significantly reduced in both SERCA2a and wild-type banded animals. Banded transgenic rats also revealed a significant increase in Tau (Fig. 5B). This may be explained by a decrease in SERCA2a protein concentration in cardiac muscle after pressure overload [3]. In order to analyze a potential correlation between Tau and the SERCA2a protein concentration in individual animals, SERCA2a protein expression in left ventricular myocardium was quantified by RIA. This method allows a precise measurement of SERCA2a in individual samples [24]. On average, banding resulted in a significant decrease in SERCA2a protein concentration compared to sham operated controls (Fig. 5A) (64.7±4.1 vs. 79.1±5.0 pmol/mg protein; P<0.05). Plotting SERCA2a protein levels against the corresponding Tau of individual transgenic hearts indicated a direct correlation (r = –0.83) between SERCA2a protein levels and Tau in both banded and sham-operated transgenic rats (Fig. 5C).


Figure 5
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  		Fig. 5 Correlation between relative SERCA2a protein concentrations and Tau in banded and sham operated transgenic rat hearts. (A) SERCA2a protein levels [pmol/mg protein] and (B) Tau in banded transgenic SERCA2a rats compared to sham operated controls. *P<0.05. (C) Total amount of SERCA2a protein determined by radioimmunoassay was related to total amount of protein. Each value was blotted against the corresponding Tau of a banded (n = 7) or sham operated (n = 6) heart from transgenic line 104.1. Increasing SERCA2a concentrations were related to a decrease in Tau. The inverse correlation (r = –0.83) was significantly different from the zero hypothesis ({alpha} = 0.05). There was no difference in the regression between banded and sham operated animals.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Two independent transgenic rat lines were established with a 30–70% overexpression of SERCA2a protein in the myocardium. Elevated SERCA2a levels were associated with shortened intracellular calcium transients, accelerated relaxation, and increased contraction parameters. Both transgenic lines revealed an attenuated negative force–frequency relation. The effect of β-adrenergic stimulation was present in a dose-dependent manner. Banding of the abdominal aorta of wild-type and high expressing SERCA2a line resulted in myocardial hypertrophy associated with reduced SERCA2a levels, lowered left ventricular pressure, as well as worsened relaxation and contraction parameters. However, SERCA2a overexpressing hearts revealed improved hemodynamic parameters in comparison to banded wild-type controls. Acceleration of isovolumetric relaxation characterized by the index Tau was directly correlated with SERCA2a protein concentration in unchallenged and banded transgenic rats suggesting SERCA2a to be one of the key players of the active process of relaxation.

4.1 Models of SERCA2a overexpression
In the past few years, transgenic mouse lines have been established by independent investigators with 20–54% overexpression of the rat SERCA2a cDNA [7,8]. In our two transgenic rat lines 104.2 and 104.1, expression of SERCA2a protein was elevated by 30–70% reflecting the high transcriptional activity of the CMV-enhanced MLC-2v promoter construct previously characterized in an in vitro differentiation model of murine embryonal stem cells [28] and used for the generation of several transgenic mouse and rat models [17,29].

In the transgenic mouse line reported by He et al., an unspecific, CMV-enhanced chicken β-actin promoter resulted in 20% overexpression of SERCA2a [7]. In the other transgenic mouse model, use of the β-myosin heavy chain promoter resulted in up to 54% overexpression of SERCA2a [8]. In concordance with our findings, baseline contraction and relaxation parameters were accelerated in these mouse lines. Similar to the transgenic mouse models, SERCA2a rats showed shortening of the intracellular calcium decline suggesting a significant effect of SERCA2a overexpression on accelerated relaxation. This can be explained by an increased diastolic calcium removal into the sarcoplasmic reticulum [8]. A further common characteristic of the transgenic mice from Baker et al. and our rat lines is increased intraventricular pressures measured in isolated working heart preparations [8]. This might be explained be an elevated calcium concentration in the SR resulting in an increased calcium efflux during systole.

Moreover, the transgenic mice by Baker et al. showed a more pronounced improvement in hemodynamic parameters in the mouse line with higher SERCA2a protein levels. This dose-dependent relationship between SERCA2a protein concentration and hemodynamics was also observed in our rat lines. However, the effect of overexpression of rat cDNA was more pronounced in mice, in which 20% overexpression resulted in a significant acceleration of relaxation parameters [7], while our transgenic rat line with 30% upregulation showed a trend towards improved relaxation. The requirement of a certain level of overexpression may also explain the lack of significant alterations in left-ventricular hemodynamic parameters in another transgenic rat model with 26% overexpression of SERCA2a [30]. However, analysis of papillary muscle preparations revealed an accelerated relaxation as in our study. Overexpression of SERCA2a by 26% was sufficient to attenuate the depressed calcium uptake in a model of diabetic cardiomyopathy [30].

4.2 Hemodynamic effects of SERCA2a overexpression
SERCA2a overexpression resulted in accelerated relaxation parameters both in isolated right ventricular papillary muscles and the working heart model. Although contraction parameters were increased in the working heart model, differences in maximum isometric force and TTP measured in isolated papillary muscles were not significant. This might be either due to different experimental approaches or regional differences in transgene expression, since papillary muscles were derived from right ventricular origin.

Increasing stimulation frequencies resulted in decreased intraventricular pressures in both transgenic rat and wild-type controls. However, SERCA2a overexpressing hearts revealed significant differences in the active phase of relaxation, which is commonly assessed by the time constant (Tau) of the exponential ventricular pressure decline occurring during the isovolumetric period of relaxation. As reported previously, wild-type hearts showed an increase in Tau with increasing stimulatory frequency [31]. In contrast, increasing heart rates did not affect Tau in transgenic hearts, indicating that the isovolumetric phase of relaxation also remains accelerated with increasing frequency. These findings may be explained by SERCA2a overexpression resulting in an increased pump activity of the cytoplasmic Ca2+ into the SR during active relaxation. An increased ability of the SR to store calcium has already been shown in right ventricular trabeculae of transgenic mice with SERCA2a overexpression [8,32]. Alignment of the Tau values with SERCA2a protein levels of individual transgenic rats revealed a direct correlation indicating a central role of SERCA2a levels in determining the isovolumetric phase of relaxation.

Activity of SERCA2a is under the control of phospholamban. In its dephosphorylated state, phospholamban binds to SERCA2a and inhibits its activity. Phosphorylation of phospholamban mediated by cAMP-dependent protein kinase and the resulting increase in SERCA2a activity were shown to be responsible for the accelerated relaxation by β-adrenergic stimulation of the heart [33,34]. Therefore, we assessed the response of the SERCA2a overexpressing rat hearts on the β-agonist isoproterenol. Increasing doses of isoproterenol resulted in elevation of left ventricular pressure and reduction of Tau in both SERCA2a transgenic and wild-type rats. These findings indicate that SERCA2a enzyme activity in transgenic hearts remains under inhibition of phospholamban and can be further increased by β-adrenergic stimulation. In concordance with previous studies of transgenic mice and rats overexpressing SERCA2a, phospholamban protein levels were not different from wild-type rats in our study [7,8,30]. However, it cannot be excluded that the basal phosphorylation status of phospholamban might be decreased in order to compensate elevated SERCA2a levels. A compensatory phosphorylation of phospholamban was observed in heterozygous SERCA2a knock out mice which was considered an adaptive change for decreased SERCA2a activity [35]. Further studies are necessary to determine alterations of the phosphorylation status in our model.

Since relaxation abnormalities in heart failure or hypertrophy are attributed to reduction of SERCA2a protein expression, we analyzed the long-term effect of SERCA2a overexpression under pressure overload conditions. Constriction of the abdominal aorta between the renal arteries results in hypertension due to renal ischemia by the Goldblatt mechanism resembling hypertrophy in patients with arterial hypertension [36]. Both banded wild-type and SERCA2a transgenic animals revealed a decrease in left ventricular pressure as well as worsening of relaxation and contraction parameters. However, banded SERCA2a transgenics revealed significantly improved hemodynamic parameters in comparison to wild-type rats. Therefore, the positive inotropic effect of SERCA2a overexpression is also preserved under pressure overload conditions.

These findings are in concordance with studies of transgenic SERCA2a mice subjected to constriction of the ascending aorta [37]. Similarly to our study, pressure overload resulted in reduced contraction and increased relaxation parameters in non-transgenic mice. Banded transgenic SERCA2a mice revealed a preserved contractile function despite significant hypertrophy [37]. The mouse model also revealed a lower mortality of SERCA2a transgenic mice compared to wild-type controls after 7 weeks of aortic banding. In contrast, all banded rats survived the whole observation period of 10 weeks. These differences in mortality might be attributed to the more profound hemodynamic consequences of banding of the ascending aorta in the murine model. Similar to transgenic mice, SERCA2a overexpression did not prevent development of hypertrophy in banded rats. It is of interest that hypertrophic response is more pronounced in banded transgenic animals compared to wild-type controls. This may be due to the increased amount of SERCA2a protein. Similarly, an increase in SERCA2a levels induced by infusion of thyroid hormone was associated with an increased hypertrophic response and improved hemodynamic parameters [38]. Hypertrophy may be a long-term side effect of SERCA2a overexpression under pressure-overload conditions which might be attenuated using a mutant of SERCA2a disrupting the functional association with phospholamban (K397/400E) [39]. Transgenic mice overexpressing this high calcium affinity mutant of SERCA2a showed an attenuated hypertrophic response 1 week after constriction of the transverse aorta [39].

A further potential limitation of SERCA2a overexpression which is associated with increased ATP consumption may be an alteration of the energy metabolism. Preliminary data showed that oxygen consumption increases with SERCA2a overexpression [40]. However, our data with an oxygen consumption measured at a stimulation rate of 3 Hz showed that even at an increased stimulation rate, where we expect an increased ATP consumption, there is no difference in diastolic and developed force. Therefore SERCA2a overexpression might be beneficial for patients with heart failure since the increased economy does not decrease contractility. Moreover, we observed no differences in mortality between wild-type and transgenic rats. However, we cannot exclude an effect of the increased energy demand on survival beyond the time-frame observed.

In summary, our transgenic rat model confirms a key role of SERCA2a in calcium homeostasis and contractile function. The effect of SERCA2a overexpression on enhancing cardiac contraction and relaxation parameters under physiological and pathological conditions in rats suggests a potential therapeutic role of SERCA2a overexpression in heart failure. However, in respect to the discouraging clinical results with positive inotropic agents such as catecholamines or phosphodiesterase inhibitors in heart failure therapy, further long-term analyses of SERCA2a overexpression will be of decisive importance prior to considering SERCA2a for gene therapy approaches in patients.

Time for primary review 26 days.


   Acknowledgements
 
We are grateful to K. Boheler, MD, Laboratory of Cardiovascular Science, National Institute on Aging, Baltimore, MD, USA, for providing the SERCA2a cDNA and F. Wuytack, MD, Laboratory of Physiology, K.U. Leuven, Belgium, for the SERCA2a antibody. We also would like to thank Y. Müller and S. Olsson for excellent technical assistance. We further thank L. Maier for critically reading the manuscript. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to W.M.F. (SFB 320/B-6).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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